What happens to PRIMERS at 72 C ? - (Jul/19/2006 )

The question may sound very stupid or trivial to an expert. But I am NOT a proffesional in molecular biology & would be glad if some one could clarify my doubt.

I found this description for the Primer Tm on one of the sites on the internet. It says:

" The melting temperature of a primer is defined as the temperature below which the primer will anneal to the DNA template and above which the primer will dissociate (break apart) from the DNA template. "

A usual cycling temp/time followed is:

Denaturation------94 C Annealing---------55 C Elongation--------72 C

In this case, the annealing temp. for the primers is 55 C. That means, it would have a "Tm" somewhere around 5 C MORE than the "Ta(opt)" i.e. around 60 C (55 + 5).

If primers denature from their target strand at ABOVE 60 C (which is their Tm), then how primers remain bound to the target at 72 C to prime the elongation step (which is carried out at 72 C) ?

Thank you !

-owl-

The answer is really to recognize 2 facts:

1) longer oligos have a higher Tm than shorter oligos2) Taq polymerase is most active at 72 degrees, although it can polymerize nucleotides at temperatures below that.

When the primer anneals in the annealing cycle, it can serve as a template for DNA amplification. The extension from this primer doesn't only happen only at 72 degrees, and by the time the extension step is reached the new oligo is long enough to remain bound to the template DNA.

-Elias-

I usually design my primers to have a Tm greater than 72 so they won't come off. Otherwise you may have to anneal at a lower temp, which causes specificity problems.

QUOTE (owl @ Jul 19 2006, 01:20 PM)

The question may sound very stupid or trivial to an expert. But I am NOT a proffesional in molecular biology & would be glad if some one could clarify my doubt.

I found this description for the Primer Tm on one of the sites on the internet. It says:

" The melting temperature of a primer is defined as the temperature below which the primer will anneal to the DNA template and above which the primer will dissociate (break apart) from the DNA template. "

A usual cycling temp/time followed is:

Denaturation------94 C Annealing---------55 C Elongation--------72 C

In this case, the annealing temp. for the primers is 55 C. That means, it would have a "Tm" somewhere around 5 C MORE than the "Ta(opt)" i.e. around 60 C (55 + 5).

If primers denature from their target strand at ABOVE 60 C (which is their Tm), then how primers remain bound to the target at 72 C to prime the elongation step (which is carried out at 72 C) ?

Thank you !

-Moz-

hithe Tm of a primer represents the temperature at which 50%of them would anneal. So increasing temp will reduce the percentage of primer annealed but it still remain enough primers to do the pcr

-fred_33-

By the time the thermal cycler has ramped up to 72C, the polymerase has had time to extend the primer and thus increase its Tm.

-tfitzwater-

Sometimes annealing temps are set up to 10 degress below the theoretical Tm, so this would give you a Tm of 65. This also is near the temperature at which Taq starts to extend a template.